In our copending applications PCT/AU/00365, PCT/AU/00723, and PCT/AU/00724 (the disclosures of which are incorporated herein by reference) we have described a method for determining the concentration of an analyte in a carrier. In that method a sample to be analysed is brought into contact with a reagent containing an enzyme and a redox mediator in an electrochemical cell. The cell is a thin layer cell comprising a working electrode spaced apart from a counter electrode by a spacer which ensures that the two electrodes have substantially identical area and predetermined spacing. The spacing between the electrodes is essentially close so that after a potential is applied between the electrodes, reaction products from the counter electrode migrate to the working electrode and vice versa, eventually establishing a steady state concentration profile between the electrodes which in turn results in a steady state current.
It has been found that by comparing a measure of the steady state current with the time rate at which the current varies in the current transient before the steady state is achieved, the diffusion coefficient of the redox mediator can be measured as well as its concentration. It can be shown that over a restricted time range a plot a ln(i/iss−1) vs time (measured in seconds) is linear and has a slope (denoted by S) which is equal to −4 p2D/L, where “i” is the current at time “t”, “iss” is the steady state current, “D” is the diffusion coefficient in cm2/sec, “L” is the distance between the electrodes in cm and “p” is the constant pi, approximately 3.14159. The concentration of reduced mediator present when the potential was applied between the electrodes is given by −2 p2iss/FALS, where “F” is Faraday's constant, A is the working electrode area and the other symbols are as given above. As this later formula uses S it includes the measured value of the diffusion coefficient.
Since L and the electrode area are constants for a given cell, measurement of i as a function of time and iss enable the value of the diffusion coefficient of the redox mediator to be calculated and the concentration of the analyte to be determined. In our copending application PCT/AU/00724 there are described methods suitable for mass production of cells having a substantially constant electrode separation L and electrode area A.
Currently glucose in blood samples is measured in pathology laboratories and the like by means of apparatus such the YSI blood analyser in which successive samples are analysed by means of a hollow cylindrical probe in which is mounted a silver and a platinum electrode. The face of the probe is fitted with a three layer membrane. The middle layer contains an immobilized enzyme which is sandwiched between a cellulose acetate and a polycarbonate membrane. The face of the probe, covered by the membrane, is situated in a buffer filled sample chamber into which successive samples are injected. Some of the sample diffuses through the membrane. When it contacts the immobilised oxidase enzyme it is rapidly oxidised producing hydrogen peroxide, the glucose forming a glucono-delta-lactone.
The hydrogen peroxide is in turn oxidised at the platinum anode producing electrons. A dynamic equilibrium is achieved when the rate of peroxide production and removal reach a steady state. The electron flow is linearly proportioned to the steady state peroxide concentration and therefore to the concentration of the glucose.
The platinum electrode is held at an anodic potential and is capable of oxidising many substances other than hydrogen peroxide. To prevent these reducing agents from contribution to sensor current, the membrane contains an inner layer consisting of a very thin film of cellulose acetate. This film readily passes hydrogen peroxide but excludes chemical compounds with molecular weights above approximately 200. The acetate film also protects the platinum surface from proteins, detergents, and other substances that could foul it. However the cellulose acetate film can be penetrated by compounds such as hydrogen sulphide, low molecular weight mercaptans, hydroxylamines, hydrozines, phenols and analytes.
In use, the sample (or a calibration standard) is dispensed in to the chamber, diluted into 600 microliters of buffer, and then a measurement is made by the probe. The sensor response increases and then reaches a plateau when a steady state is reached. After several seconds a buffer pump flushes the chamber and the sensor response decreases.
The apparatus monitors the base line current. If it is unstable a buffer pump will continue to flush the sample chamber with buffer. When a stable base line is established an automatic calibration is initiated. The apparatus calibrates itself for example after every five samples or 15 minutes. If a difference of more than 2% occurs between the present and previous calibration, the apparatus repeats the calibration. Recalibration also occurs if the sample chamber temperature drifts by more than 1° C.
The apparatus described suffers from a number of disadvantages. Firstly, a high proportion of its time in use is spent in performing calibrations rather than analysis. Furthermore the consumption of buffer and calibrating solutions is a substantial cost. Another disadvantage is that as the enzyme membrane ages, a graph of reading versus concentration becomes non-linear. It would be highly desirable to provide apparatus which is able to make measurements of the kind described with improved speed, efficiency, and at lower running cost.